Method for screening crystallization conditions in solution crystal growth

ABSTRACT

A method of screening protein crystal growth conditions with picogram to microgram amounts of protein in picoliter or nanoliter volumes is provided. A preferred method comprises a microarray with a plurality of micro-chambers in the microarray. A protein solution is placed into the micro-chambers by an automated dispensing mechanism. The protein crystal growth conditions of each of the micro-chambers is adjusted so that the protein crystal growth conditions in at least two of the micro-chambers differs. Crystallization of the protein solution in the micro-chambers is effected. For example, crystallization can be effected by a precipitate solution and/or placing an oil barrier over the protein solution. Protein crystal growth in the micro-chambers is then observed.

[0001] The present invention is a continuation-in-part of U.S. Ser. No.09/543,326, filed Apr. 5, 2000, which claims priority to from U.S. Ser.No. 60/128,018, filed Apr. 6, 1999, both hereby expressly incorporatedby reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the crystallization of proteinsin or from protein solutions. The present invention particularly relatesto a method of screening a large number of protein crystal growthconditions which may be conducive to protein crystallization. Even moreparticularly, the present invention relates to a method which identifiesone or more optimum protein crystal growth conditions, while at the sametime using substantially less protein solution.

BACKGROUND OF THE INVENTION

[0003] The crystallization of proteins for structure-function studiesand structure based drug design has become an increasingly importantpart of biotechnology research. When crystal growth is attempted for anew protein, the appropriate chemical conditions (i.e. proteinconcentration in solution, precipitate type and concentration, pH, andgrowth temperature) are unknown and have typically been determined bytrial and error experimentation.

[0004] Typically 1000 or more different sets of crystal growthconditions are screened to determine conditions conducive tocrystallization. The screening involves repetitive procedures that areextremely laborious and tedious. With present laboratory protein crystalgrowth equipment, each crystallization chamber requires about onemicro-liter of protein solution. The protein solutions typically haveconcentrations in the range of 10 to 25 micrograms per microliter tofacilitate crystal growth. Therefore, to screen 1000 samples typicallyrequires between 10 and 25 milligrams of protein. This is a considerableand costly amount, especially for proteins that are difficult to isolateor generally express. A large percentage (about 50%) of the proteinscannot easily be expressed in milligram quantities.

[0005] Thus, it would be desirable to provide methods for screeningprotein crystal growth conditions that require picogram to microgramamounts of protein for each screening condition. Preferably such methodswould require only picogram to nanogram amounts of protein in picoliterto nanoliter volumes in each screening condition sample.

[0006] It would be further desirable to provide high throughputscreening methods for screening protein crystal growth conditions in alarge number of samples on a sub-microgram scale. These methods woulduse a microarray as a platform for protein crystal growth. The methodswould also utilize automatic dispensing of solutions and scoring ofcrystal growth.

SUMMARY OF THE INVENTION

[0007] The present invention is a method of screening protein crystalgrowth conditions employing a minimal amount of protein, preferably on apicogram to microgram scale. Each screening sample has picogram tomicrogram amounts of protein in a picoliter to nanoliter volume.Predetermined protein crystal growth conditions are maintained andcrystal growth is analyzed using both qualitative and quantitativecriteria.

[0008] In a preferred embodiment, a microarray is provided for use inmethods of screening protein crystal growth. Preferably the microarrayhas a plurality of micro-chambers in the microarray. The micro-chambersmay be passive or a combination of passive micro-chambers that areconnected with miniaturized active control elements such as, but notlimited to, valves, pumps and electrodes. A protein solution isautomatically dispensed into the micro-chambers. The protein crystalgrowth conditions of each of the micro-chambers is adjusted so that theprotein crystal growth conditions of at least two of the micro-chambersdiffers. Protein crystal growth in the micro-chambers is then analyzedbased on both the qualitative amount of crystallization and the qualityof the crystals formed.

[0009] Additional objects, advantages, and features of the presentinvention will become apparent from the following description andappended claims, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The various advantages of the present invention will becomeapparent to one skilled in the art by reading the followingspecification and subjoined claims and by referencing the followingdrawings in which:

[0011] FIG. 1 is a schematic illustrating a two well design in amicroarray;

[0012] FIG. 2A is a schematic showing a top view of the placement ofprotein and precipitate solutions in a one well design;

[0013] FIG. 2B is a schematic showing a side view of placement ofprotein and precipitate solutions in a one well design;

[0014] FIG. 2C is a schematic showing a side view of an alternativeplacement of protein and precipitate solutions in one well;

[0015] FIG. 2D is a schematic showing a side view of placement ofprotein and precipitate solutions in two wells;

[0016] FIG. 2E is a schematic showing a top view of the placement ofprotein and precipitate solutions in a two well design;

[0017] FIG. 3 is a photograph showing a microarray; and

[0018] FIG. 4 is a photograph of a protein crystal obtained withnanogram amounts of protein in nanoliter volumes.

DETAILED DESCRIPTION OF INVENTION

[0019] The method of the present invention is for screening proteincrystal growth conditions in protein solutions employing a minimalamount of protein in a minimal volume, preferably on a pico, nano ormeso scale. Pico, nano or meso scale as used herein preferably employs(on average) picogram (pg), nanogram (ng) or microgram (μg) amounts ofprotein in picoliter (pl) or nanoliter (nl) volumes. Preferably, theamount of protein in each screening sample is less than about 5 μg. Morepreferably, the amount of protein in a screening sample will be lessthan about 1 μg. In one embodiment, the volume of protein solution in ascreening sample is preferably from about 0.001 nl to about 250 nl andmore preferably about 0.01 nl to about 10 nl. It will be appreciated bythose skilled in the art that the volumes actually employed for anyparticular protein will be a function of (without limitation) the targetprotein and its concentration in the protein solution.

[0020] The protein solution contains one or more desired proteins forcrystallization. As used herein, the term “protein” is meant to includeall naturally occurring and synthetic peptides, polypeptides, proteins,and protein complexes. In one preferred embodiment the concentration ofprotein in the solution is from about 0.1 μg/μl to about 50 μg/μl, morepreferably from about 0.1 μg/μl to about 10 μg/μl, and still morepreferably about 0.1 μg/μl to about 1.0 μg/μl. In another preferredembodiment, the solution is buffered to a pH between about 2.0 and about10.0, more preferably from about 3.0 to about 8.5. If desired, theprotein solution may optionally contain agents that aid in solubilizingthe protein at the desired protein concentration or conditions. Forexample, if the protein is a membrane protein, the protein solution mayoptionally contain one or more surface active agents, such as adetergent mixture. In one preferred embodiment, the protein solutionalso comprises components that assist in crystallization. By way ofnon-limiting example, the protein solution will comprise an aqueous saltsolution, polyethylene glycol, or an alcohol. Such components as well astheir selection, ranges, contraindications and the like are well knownto those skilled in the art. See, for example, Gilliland, G. L. et al.,Acta Crystallogr. D50:408-413 (1994); McPherson, A., Crystallization ofBiological Molecules, Cold Spring Harbor Press, Cold Spring Harbor,N.Y., pp. 487-524 (1999), expressly incorporated by reference.

[0021] The protein solution is dispensed onto a platform. The platformcan be, by way of non-limiting example, a glass slide, a multi-wellplate or a microarray. The solution is preferably dispensed using adevice with picoliter or nanoliter accuracy. Preferably the dispensingdevice has at least a 90% accuracy on a picoliter or nanoliter scale.The protein solution can be dispensed manually using, for example, asyringe. In a highly preferred embodiment, automatic dispensing devicesare used to dispense the protein solution.

[0022] A second solution, the reservoir or precipitate solution isprovided. The precipitate solution is a solution that helps to bringabout protein crystallization. It can comprise, for example, a saltsolution, an alcohol or a polyethylene glycol. The second solution isprovided either before, after, or simultaneously with the proteinsolution. The volume of the precipitate solution is typically equal toor greater than the volume of protein solution. The placement of thesecond solution is dependent on the crystallization method used but istypically in fluid communication with the first solution. Fluidcommunication can be liquid-liquid, liquid-vapor or vapor-vaporcommunication. Generally, a channel is provided for fluid communication.A channel is broadly defined herein as a space that enables fluidcommunication to occur. In the liquid-liquid diffusion method, theprotein solution and precipitate solution contact each other at aninterface. In batch crystallization, the two solutions are mixedtogether. If vapor diffusion crystallization is desired, the twosolutions are kept separate but space is allowed for the diffusion ofvapor between the solutions. Or, in an alternate embodiment, a singlesource or reservoir of the second solution may be employed. In yetanother alternate embodiment, a desiccant source or a dry gaseousreservoir may be employed in place of the second solution. Specificconditions and variations in these methods are well known to the skilledartisan.

[0023] Protein crystal growth is monitored periodically, eitherqualitatively, quantitatively, or both. This may be by manual inspectionusing high resolution microscopy or electron microscopy. Preferably,protein crystal growth may be monitored automatically, by, for example,high resolution optical means which automatically detects crystal growthbased on, for example, edge analysis. If desirable crystal growth isobserved in a sample, the protein crystal growth conditions of thatsample can be reproduced on a macro scale to produce a protein crystalfor further analysis. Alternatively, if a precipitate or a clear sampleis observed, these conditions can be used to optimize the conditions foradditional screening. It will be appreciated that the platform mustemploy at least one path that is visually and/or optically clear to themethod of detection.

[0024] In at least one preferred embodiment the method of the presentinvention for screening protein crystal growth employs a microarray witha plurality of wells or reservoirs as the platform. A microarray may beconstructed, for example, similar to a micro-electromechanical chip. Themicroarray preferably has a planar shape and employs a size andthickness that are compatible with manual or automated plate grippers.The microarray can be made from different materials and by differenttechniques known to those skilled in the art. The material of themicroarray that includes the wells or reservoirs is preferably minimallywater absorbing, and is otherwise sufficiently unreactive with thecomponents of the solution. This may be done as a laminate or providedas a coating, for example. Alternatively, a material that absorbs waterat a predictable rate can also be used to construct the wells orreservoirs. The volumes of protein and precipitate solutions may then beadjusted to compensate for the water absorption of the material.Preferred materials include, but are not limited to, glass, fusedsilicon, quartz, a silicon wafer, a polymer or a polysulphone.Alternatively, the microarray can be made from a material coated with ahydrophobic material, such as polysulphone, to limit water absorption inthe microarray. Alternatively, the microarray comprises more than onematerial. Preferably, the microarray is a composite with a bottom ofthin glass plate bonded to plastic, glass, silicon rubber or othermaterials in which wells can be manufactured, with at least one sideproviding an optical path that is acceptable to the detection techniqueemployed.

[0025] In an alternate embodiment, the surfaces of the wells arehydrophobic. For example, the material of the microarray may have ahydrophobic surface. Alternatively, the surfaces of the wells may becoated with a hydrophobic coating. Although not necessary, thehydrophobic surfaces of the wells prevent the drops of solutions fromspreading.

[0026] The microarray includes a multitude of micron sized wells on thesurface of the chip. The term wells encompasses wells, micro-chambersand any indentation sufficient of holding or retaining a desired volumeof from about 0.001 nl to about 500 nl, preferably from about 0.01 nl toabout 20 nl. The wells are spaced from each other on the surface. Theprecise number of wells on the surface of the microarray can vary, andthe total number of wells on the surface is a matter of choice for theuser.

[0027] Each of the wells has a volume sufficient to hold an amount ofprotein solution adequate for growing a protein crystal. Preferably,each of the wells holds a volume from about 0.001 nl to about 250 nl,preferably from about 0.01 nl to about 10 nl.

[0028] The wells of the microarray are made by using an etchant such ashydrogen fluoride or by other known etching or fabrication techniques.

[0029] The wells can include known means for controlling conditions,individually or collectively, such as pressure, heating or cooling thewells, humidity levels in the wells as well as known means for stirringmaterials loaded into the wells.

[0030] In one arrangement, the wells of the microarray are not connectedand separate from each other. In an alternative arrangement, adjacentwells of the microarray are connected by one or more channels whichprovide fluid communication between the adjacent wells (FIGS. 1 and2D-E). Preferably, the connecting channels will have cross-sectiondimensions and length allowing control over the rate of transport offluid, vapor, buffer, or precipitating or crystallizing agents throughthe channels. In one embodiment, varying the dimensions of the channelscontrols protein crystal growth condition. In an alternate embodiment,protein crystal growth conditions are controlled by placing a materialin the micro-channels that controls fluid communication between thewells. Non-limiting examples are membranes, acrylamide or agarose. Forexample, the connecting micro-channels are from about 0.0001 to about0.2 microns wide and from about 0.00005 to about 0.1 microns deep.Alternatively, the micro-channels are from about 0.0001 to about 2.0microns wide and from about 0.00005 to about 0.5 microns deep. Themicro-channels are formed in the microarray chip by the known etchingtechniques.

[0031] An example of two wells in a microarray (10) connected by amicrochannel is shown in FIG. 1. The protein solution well 12 isconnected to precipitate solution well 14 by a microchannel 16. Thedimensions of each well are designed to hold the desired amount ofsolution and may have the same or different dimensions. Initially,protein sample is dispensed into well 12 to an initial liquid height 18and precipitate solution is dispensed into well 14 with liquid height20. The top of the wells and microchannel are sealed by an opticallyclear cover 22. In vapor diffusion crystallization, the precipitatesolution in well 14 has a lower vapor pressure than the protein solutionin well 12, causing diffusion of solvent from well 12 to well 14 untilthe solution liquid height in well 12 reaches a final height 24. Theconcentration of the protein solution in well 12 precipitates proteincrystal formation.

[0032] The microarray can also include a known means for transmitting afluid or gas to the wells of the microarray from an external source. Forexample, an external mechanical pumping system marketed byWatson-Marlowe, Inc., under the trade designation “205U” can be used.The pumping system is a multi-channel cassette which delivers fluid orgas in reproducible and accurately controlled amounts.

[0033] Optionally, micro-valves are disposed in the wells andmicro-channels to regulate the flow of fluid or vapor between the wellsand through the microchannels in a known manner.

[0034] An automated dispensing mechanism capable of accurately and/orrepeatedly dispensing picoliter and/or nanoliter volumes is alsoprovided. Preferably, the automated dispensing mechanism has an accuracyof at least about 90%. The automated dispensing mechanisms arepreferably Piezo-based or fast solenoid dispensing mechanisms. Morepreferable, the dispensing mechanism is a fast solenoid dispensingmechanism. The dispenser has a large number of parallel capillaries. Thecapillaries are in fluid communication with a source of proteinsolution, a source of precipitate solution, and a source of buffersolution. The dispensing can be actuated by ultrasonic transducers thatefficiently produce a pressure wave in the capillaries that contain thesolutions. The dispenser is analogous to ink jet printer heads forcomputer printers but the fluid is not heated, thus not damaging thesolutions.

[0035] The protein solution preferably comprises an aqueous proteinsolution at a concentration of from about 0.1 μg/μl to about 50 μg/μl.Preferably, the concentration is from about 0.1 μg/μl to about 10 μg/μl,more preferably from about 0.1 μg/μl to about 1.0 μg/μl. Preferably, theprotein solution comprises a detergent mixture when crystallizingmembrane proteins. The precipitate solution preferably comprises aconcentrated aqueous salt solution or polyethylene glycol asprecipitating agents. The buffer solution preferably has pH betweenabout 2 and about 10.

[0036] The automated dispensing mechanism dispenses an initial volume ofprotein solution, an initial volume of precipitate solution, and aninitial volume of buffer solution from the source of protein solution,the source of precipitate solution, and the source of buffer solution,respectively, into preselected wells or connecting channels of themicroarray.

[0037] The placement of the initial volume of protein solution, theinitial volume of precipitate solution, and the initial volume of buffersolution in the preselected wells or channels of the microarray isdependent upon the method utilized to effect crystallization of theprotein in the protein solution.

[0038] Preferred methods to effect crystallization of the protein in theprotein solution include liquid-liquid diffusion, batch diffusion, andvapor diffusion.

[0039] In the liquid-liquid diffusion method, the initial volume ofprotein solution is placed in one set of preselected wells, and theinitial volume of precipitate solution is placed in a separate ordifferent set of wells. The protein solution wells are connected to theprecipitate solution wells by micro-channels. The initial volume ofbuffer solution may be placed in the micro-channels, or alternativelyadded directly to the initial volume of protein solution and/orprecipitate solution.

[0040] The concentration, amounts, precipitate type, and pH of theinitial volumes of protein solution, precipitate solution, and buffersolution are primary conditions which determine protein crystal growthin a protein solution. In preparing the initial solutions, and in theautomated dispensing mechanism placement, these conditions and thesample placement are varied in accordance with a pre-designed program.

[0041] A cover plate is affixed to the microarray to convert the wellsto micro-chambers and to convert the micro-channels to a capillary tubestructure. The cover plate can made of the same or different material asthe microarray, but the cover plate (or some portion of the well orchamber) must be transparent to permit optical analysis of the proteinsolutions in the chambers of the microarray. Preferably, the cover platewill be glass or other material that is visually or optically clear,such as an optically clear tape.

[0042] Alternatively, the enviromnent surrounding the microarray can becontrolled to limit evaporation of the solutions. Under controlledconditions of, for example, temperature and humidity, covering thesamples may not be necessary.

[0043] The crystallizing agent in the precipitate solution, in selectedmicro-chambers, diffuses via the connecting capillaries to selectedmicro-chambers containing protein solution.

[0044] Protein crystal growth in the different chambers are thenmonitored by high resolution or other optical means which automaticallydetects crystal growth based on well known edge analysis. Alternatively,the protein crystal growth can be monitored by manual inspection usinghigh resolution microscopy or electron microscopy. Preferably theprotein crystal growth in the chambers is monitored by high resolutionoptical means which automatically detects crystal growth based on edgeanalysis.

[0045] Once crystal growth in a chamber is detected, that chamber'sprotein crystal growth conditions can be reproduced on a macro scale toproduce a protein crystal which can be analyzed by x-raycrystallography. Alternatively, if a precipitate or clear sample isobserved, the conditions in those samples can be used to optimizeconditions for additional screening.

[0046] In the vapor diffusion method, the initial volume of proteinsolution is placed in one set of preselected wells, and the initialvolume of precipitate solution is placed in a separate or different setof wells based on a pre-designed program, as with the liquid-liquiddiffusion method (FIGS. 2D-E). The protein solution wells are connectedto the precipitate solution wells by micro-channels. The initial volumeof buffer solution is added to the initial volume of protein solutionand/or initial volume of precipitate solution. Alternatively, theprotein solution and precipitate solution can be placed in the same wellsuch that the two solutions do not come into contact (FIGS. 2A-C).

[0047] As with liquid-liquid diffusion, the crystal growth is varied indifferent wells in accordance with a pre-designed program in which theplacement, concentration, amounts, precipitate type, and pH conditionsare varied in the different wells.

[0048] A cover plate is then affixed to the microarray as with theliquid-liquid diffusion method. The vapor pressure of the precipitatesolution is lower than the vapor pressure of the protein solution. Thiscauses the protein solution in a micro-chamber which is connected via acapillary to a micro-chamber containing a precipitate solution toevaporate and become super-saturated causing precipitation of protein.Crystal growth is monitored as in the liquid-liquid diffusion.

[0049] Alternatively, the protein solution is placed into wells of themicroarray and the microarray is exposed to a single reservoir with theprecipitate solution. This method allows for less fluid dispensing, butalso less control of the protein crystal growth conditions with respectto each protein sample.

[0050] To further control protein crystal growth the protein solutionand/or precipitate solution can be covered with an oil solution.Preferably, the oil solution is inert and immiscible with respect to theprotein and precipitate solutions while allowing for water diffusionthrough the oil and has a density less than the protein and/orprecipitate solution. More preferably, the oil solution containsparaffin oil, silicone oil, or mixtures thereof and most preferably, theoil contains a mixture of paraffin and silicone oils. It will beappreciated by the skilled artisan that other oil mixtures may be usedas long as the oils are miscible with each other. See Chayen, N. E.,Structure 5,1269-1274 (1977).

[0051] Water can evaporate at different rates through different oils.Paraffin oil can act as a good sealant allowing only a negligable amountof water evaporation through it during the average time required for acrystallization experiment. In contrast, water can diffuse readilythrough silicone oils. A mixture of paraffin and silicone oils permitspartial diffusion, with the amount and rate of diffusion controlled bythe ratio at which they are mixed. If a mixture of paraffin and siliconeoils are used, the ratio of paraffin oil to silicone oil preferably isfrom about 90:10 vol % to about 10:90 vol %. More preferably, the ratiois from about 90:10 vol % to about 50:50 vol % paraffin oil to siliconeoil. It will be appreciated that the preferred ratio of paraffin oil tosilicone oil will also depend on whether the wells are further sealedwith a coverplate to form discrete microchambers in each well. When acoverplate is used evaporation is limited and a higher ratio of siliconeoil may be used. However, when all the wells of the microarray areexposed to a single reservoir of precipitate solution, use of a coverplate may not be feasible and it may be desirable to have a higherproportion of paraffin oil in the oil mixture.

[0052] Alternatively, covering the protein and/or precipitate solutionwith at least two distinct layers of oil can control protein crystalgrowth. Preferably both oils are inert and immiscible with respect tothe protein and/or precipitate solutions and have a density less thanthe solutions. More preferably the oils have different densities suchthat they form discrete layers, one on top of the other, covering theprotein solution. The oils may be either miscible or immiscible withrespect to the oils. It will be appreciated that if the oils aremiscible, additional care will be taken addition of the oils to thewell. Most preferably, the oils also have different water diffusionproperties with respect to each other. By way of non-limiting example, alayer of paraffin oil having a density heavier than silicone oil can beadded to a well containing protein solution. A layer of silicone oil isthen carefully added on top of the paraffin oil layer. It will beappreciated that crystal growth conditions can be further controlled byadjusting the volume of each oil layer covering the solutions.

[0053] Protein crystallization can also be controlled by the thicknessof the oil layer, which in practice is controlled by the volume. Alarger volume of oil in relation to the volume of the protein and/orprecipitate solution will result in slower vapor diffusion. In contrast,the smaller the volume of oil, the vapor diffusion will be more rapid.Preferably the volume of oil in each well is from about 1 ml to about100 ml and more preferably from about 10 ml to about 80 ml.

[0054] In one method of the present invention, the drop of protein orprecipitate solution is placed in the well initially and then the oilsolution is added to the well. In an alternate method, the oil may beadded to the well first and then the protein or precipitate solution isdispensed directly into the oil solution. With either method the proteinor precipitate solution, having a density greater than the oil, will bepositioned on the bottom of the well.

[0055] A further method of the present invention includes “containerlesscrystallization” in which a drop of protein solution is positionedbetween two layers of two different oils. The protein solution thereforedoes not come in contact with the sides of the well. Preferably the twooils are immiscible with respect to each other and the protein solution.More preferably a flat meniscus forms between the two oil layers. Thebottom oil will have a higher density than the protein solution whilethe top oil will have a lower density than the protein solution.Preferably the bottom oil is a high density fluorinated silicone oil andthe top oil is a silicone oil. In a preferred method, both oils areadded to a well. They can be added simultaneously or sequentially. Adrop of protein solution is then added to the well. The drop of proteinsolution should be positioned between the two oil layers toward thecenter of the well and not be in contact with the sides of the well. SeeChayen, N. E., Protein Engineering 9, 927-929 (1996).

[0056] In the batch method, the volume of protein solution, the volumeof precipitate solution, and the volume of buffer solution are placedtogether in individual wells of the microarray. In this method, the chipdoes not have connecting channels between the wells.

[0057] As with liquid-liquid diffusion and vapor diffusion methods, thecrystal growth is varied in different wells in accordance with apre-designed program in which the placement, concentration, amounts,precipitate type, and pH conditions are varied in the different wells.

[0058] As with liquid-liquid diffusion and vapor diffusion methods, acover plate is affixed to the microarray, and the crystal growth is thenmonitored. Additionally, as described for vapor diffusion methods, theprotein and precipitate solutions may be immersed in an oil solution.

[0059] If desired, fluid or gas can delivered to the micro-chambers inreproducible and accurately controlled amounts from an external sourceby the external mechanical pumping system described above. Gas can alsobe delivered from the pressure generated by a standard glass bottle ortank. The fluid or gas delivered to the micro-chambers can be regulatedby the micro-valves. The fluid or gas can be used to further alter thecrystal growth conditions of the micro-chamber and increase the size ofthe protein crystals grown. These protein crystals can then be harvestedand examined by x-ray crystallography or nuclear magnetic spectroscopyor other appropriate techniques.

[0060] Advantages of the present invention should now be apparent. Thepresent invention provides a method of screening protein crystal growthconditions on a nano or meso scale. The method provides a means ofscreening protein crystal growth conditions for proteins that cannot beexpressed in milligram quantities as well as those that can be expressedin larger quantities. Moreover, the substantial reduction in proteinneeded for the present invention reduces the costs associated withscreening protein crystal growth conditions.

[0061] Also provided is an apparatus for screening crystal growthconditions. The apparatus comprises a microarray for the protein andprecipitate solutions, an automatic dispensing mechanism for dispensingthe solutions and an automated means for analyzing crystal growth.

[0062] The desired solutions, i.e., protein, precipitate and a buffer,are preferably automatically dispensed at a preset picoliter ornanoliter volume into the microarray by an automated dispensingmechanism. Preferably, the automatic dispensing mechanism dispensesdiscrete drops. Screening conditions such as the type of buffer and pHcan be varied from sample to sample by programming the automaticdispenser. For example, arbitrary screens varying pH could be programmedby mixing the proper ratios using different drop counts from differentstock solutions having different pH values. A pH range from 2.0 to 10.0is then screened in steps of 0.2-0.5 pH units. Other conditions, such ascrystallization agents and salt concentration are also controlled in asimilar manner.

[0063] Mixing of the reagents can either be done before dispensing orafter the solutions are dispensed into the microarray. Mixing in themicroarray, for example, can be accomplished by ultrasonic mixing,high-speed dispensing of picoliter drops, rapid temperature fluctuation,or by static diffusion.

[0064] After mixing, preferably the wells of the microarray are sealedto control the microenvironment in the wells and to prevent evaporationof the solutions to dryness. Preferably the wells are sealed with anoil, a glass cover plate, or an optically clear tape. More preferably,the wells are sealed with optically clear tape and most preferred, thewells are sealed with both oil and either a glass cover plate oroptically clear tape. The oil can be added at any time during thedispensing of the solutions. Sealing the microarray involves an armmounted on a YZ transverse mechanism. The X direction is along the platetransport direction. The arm, holding a roll of clear tape, moves pastthe last well in the row, drops to form positive vertical (Z axis)pressure, and then begins to move back in the negative Y direction whileat the same time rotating the tape roll. Once the tape leaves the platearea, a guillotine mechanism shears the tape. The plate then moves inthe X direction onto the next indexed row and the dispense processinitializes again. Automated taping is reliably performed in manyindustries.

[0065] Protein crystal growth in the different wells is monitored byhigh resolution optical means which automatically detects crystal growthbased on well known edge analysis. Such image acquisitions systems arecommercially available.

[0066] The foregoing and other aspects of the invention may be betterunderstood in connection with the following example, which is presentedfor purpose of illustration and not by way of limitation.

EXAMPLE 1

[0067] Nanoliter protein droplets were used for vapor diffusion, batchand liquid diffusion crystallization screening. The protein solutions ofeither lysozyme, thaumatin, or NAD synthetase were applied using a fivemicroliter Hamilton syringe. To ensure complete wetting of the smalldroplet to the experiment chamber, the tip of the Hamilton syringe wasplaced in contact with the wall of each experiment chamber. A variety ofmicroarrays were designed to accommodate protein solution droplets withvolume ranges of 5-20 nanoliters and precipitate volumes of 100-200nanoliters. The array prototyping was accomplished using MicroScopeslides with permanent sealing of neoprene gaskets of varying thickness(0.1 mm 0.5 mm). Once all solutions were applied to an individualexperiment chamber within the microscope slide, the experiment wassealed (with oil or grease) by placing a glass cover slide over the topof the gasket. FIG. 3 is a photograph of a typical design for a 60chamber array prototype (gasket thickness=0.1 mm) and FIG. 4 is aphotograph of crystals that were grown to 10 nanoliter protein dropletsusing a similar microarray slide.

[0068] A Cartesian robotic dispensing system was used to preparecrystallization solutions in a 6 by 10 experiment array. Five nanolitersof protein plus five nanoliters of precipitant were dispensed into onemerged droplet in one depression in the experiment chamber (FIG. 3) and50 nanoliters of precipitant plus 50 nanoliters of buffer were mergedinto one droplet in the connected depression. Thus, four solutions weredispensed for each experiment, and 6×10×4=240 total for the entire 6 by10 array. Cartesian's instrument was able to dispense all of thesolutions in less than 20 minutes. All external conditions used wereknown crystallization conditions for the particular proteins tested. Theexperiment was manually sealed and incubated at 22° C. for a period ofone day. Crystals were observed in seventy percent of the droplets.While not wishing to be bound by theory, it is believed that the failureto observe crystals in 30% of the wells was due to inaccurate dispensingof the protein and precipitant five nanoliter drops in that the peizotip did not position the drops together.

[0069] From the above description of the invention, those skilled in theart will perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims.

Having described the invention the following is claimed:
 1. A method ofscreening protein crystal growth conditions employing picogram, nanogramor to microgram amounts of protein comprising the steps of: providing amicroarray with a plurality of wells in said microarray; accuratelydispensing a volume of from about 0.001 nl to about 250 nl of a proteinsolution into said wells; accurately dispensing a volume of an oilsolution into said wells, wherein said oil solution has a density lessthan the protein solution and is inert to the protein solution;controlling the protein crystal growth conditions of each of said wellsso that the protein crystal growth conditions in at least two of saidmicro-chambers differs; and observing protein crystal growth or proteinprecipitation in said wells.
 2. The method of claim 1 wherein saidprotein solution comprises a component selected from the groupconsisting of buffers, surface active agents, salts, alcohols,polyethylene glycol and mixtures thereof.
 3. The method of claim 1wherein said protein solution is buffered.
 4. The method of claim 1wherein controlling protein crystal growth comprises employing aprecipitate solution that is in fluid communication with the microarray.5. The method of claim 1 wherein controlling protein crystal growthcomprises adding a precipitate solution to the microarray.
 6. The methodof claim 5 wherein said precipitate solution and said protein solutionare in different wells in said microarray and said microarray haschannels for fluid communication between a well comprising proteinsolution and a well comprising a precipitate solution.
 7. The method ofclaim 6 wherein controlling protein crystal growth further comprisesemploying varying dimensions of the channels.
 8. The method of claim 6wherein said precipitate solution and said protein solution are in fluidcommunication via micro-channels.
 9. The method of claim 5 wherein saidprecipitate solution and said protein solution are in fluidcommunication.
 10. The method of claim 9 wherein fluid communication isby liquid-liquid diffusion of said precipitate solution and said proteinsolution.
 11. The method of claim 9 wherein said precipitate solutionhas a lower vapor pressure than the protein solution and fluidcommunication is by vapor diffusion.
 12. The method of claim 5 whereinthe protein solution and precipitate solution are in the same well andsaid crystallization is effected by batch crystallization.
 13. Themethod of claim 12 wherein said wells further comprise a buffersolution.
 14. The method of claim 5 wherein the precipitate solution hasa volume from about 0.001 nl to about 250 nl.
 15. The method of claim 1wherein protein crystal growth or protein precipitation is observed bymicroscopy.
 16. The method of claim 15 wherein the microscopy isdifferential interference contrast microscopy.
 17. The method of claim 1wherein the protein solution is dispensed into said wells by fastsolenoid dispensing.
 18. The method of claim 1 wherein the oil solutioncomprises paraffin oil, silicone oil or mixtures thereof.
 19. The methodof claim 1 wherein the oil solution comprises a mixture of paraffin andsilicone oils, wherein the ratio of paraffin to silicone is from about90:10 vol % to about 50:50 vol %
 20. The method of claim 1 wherein thevolume of the oil solution is from about 1 μl to about 100 μl.
 21. Amethod of screening protein crystal growth conditions employing picogramto microgram amounts of protein comprising the steps of: accuratelydispensing a volume from about 0.001 nl to about 250 nl of a proteinsolution onto a platform; accurately dispensing a volume of an oilsolution onto a platform, wherein the oil solution covers the proteinsolution; controlling the protein crystal growth condition of thesample; and observing a protein precipitate or protein crystals in thesample.
 22. The method of claim 21 wherein the sample is accuratelydispensed by fast solenoid dispensing.
 23. The method of claim 21wherein controlling the protein crystal growth condition comprisesemploying a protein precipitate solution.
 24. The method of claim 23wherein said precipitate solution has a lower vapor pressure than theprotein solution and crystallization is effected by vapor diffusion. 25.The method of claim 23 wherein the protein solution and precipitatesolution mixed together and said crystallization is effected by batchcrystallization.
 26. The method of claim 23 wherein crystallization iseffected by liquid-liquid diffusion of said precipitate solution andsaid protein solution.
 27. The method of claim 21 wherein the platformis a microarray.
 28. The method of claim 21 wherein protein crystalgrowth or protein precipitation is observed by microscopy.
 29. Themethod of claim 28 wherein the microscopy is differential interferencecontrast microscopy.
 30. The method of claim 21 wherein the oil solutioncomprises paraffin oil, silicone oil or mixtures thereof.
 31. The methodof claim 21 wherein the oil solution comprises a mixture of paraffin andsilicone oils, wherein the ratio of paraffin to silicone is from about90:10 vol % to about 50:50 vol %.